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transplantation; lymphoblastic leukemia; lymphocyte count; relapse. Introduction. The therapy of childhood acute lymphoblastic leukemia (ALL) remains one of ...
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Lymphocyte recovery after allogeneic bone marrow transplantation predicts risk of relapse in acute lymphoblastic leukemia S Kumar1, MG Chen2, DA Gastineau1, MA Gertz1, DJ Inwards1, MQ Lacy1, A Tefferi1 and MR Litzow1 1 Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN, USA; and 2Division of Radiation Oncology, Mayo Clinic, Rochester, MN USA

Allogeneic blood and marrow transplantation (BMT) is curative for many patients with high-risk and relapsed acute lymphoblastic leukemia (ALL). However, relapse is an important cause of post-transplantation failure, and there are no reliable markers to predict relapse. A retrospective review of patients with ALL who underwent matched related allogeneic BMT was carried out to examine whether the rate of lymphocyte recovery after transplantation had any prognostic value in ALL. The absolute lymphocyte count (ALC) at days 21 and 30 after transplantation was obtained for 43 patients who received transplants during an 18-year period. Patients with an ALC of 175  106/l or less on day 21 were more likely to relapse than those with ALC greater than 175  106/l (relative risk, 4; 95% confidence interval, 1.5–11.2). Patients with slower lymphocyte recovery had significantly lower relapse-free survival than those with faster recovery (P ¼ 0.0028). There was also a trend toward poorer overall survival among those with a slow lymphocyte recovery (log-rank test; P ¼ 0.028). The rate of lymphocyte recovery is prognostic in patients with ALL undergoing allogeneic BMT, and this should be integrated with other predictors to identify patients at high risk of relapse. Such patients could be considered for interventions aimed at prevention of relapse, including rapid withdrawal of immunosuppressive medication or donor lymphocyte infusion. Leukemia (2003) 17, 1865–1870. doi:10.1038/sj.leu.2403055 Keywords: allogeneic transplantation; bone marrow transplantation; lymphoblastic leukemia; lymphocyte count; relapse

Introduction The therapy of childhood acute lymphoblastic leukemia (ALL) remains one of the success stories of oncology, but long-term leukemia-free survival for adults with ALL remains low (20– 40%).1 Philadelphia chromosome is present in adults at a much higher rate than in children, and older age appears to be a poor prognostic factor. The role of transplantation for patients in first remission without adverse prognostic factors remains unclear.2,3 Although transplantation certainly appears to benefit patients with relapsed disease and selected patients with high-risk disease in the first remission,4–7 20–40% of patients who have complete remission after allogeneic blood and marrow transplantation (BMT) for ALL eventually have a relapse.5,8–10 Several factors, including karyotypic abnormalities, stage of the disease, conditioning regimen used, graft-versus-host disease (GVHD) prophylaxis, and development of acute and chronic GVHD,11,12 influence the risk of relapse after allogeneic stem cell transplantation for ALL. Identification of patients at a high risk of relapse early in the post-transplantation period would allow evaluation of different prophylactic strategies. We and others have shown that slow recovery of the lymphocyte count after allogeneic BMT predicts increased risk of relapse for patients with acute myelogenous leukemia.13,14 Correspondence: MR Litzow, Division of Hematology and Internal Medicine, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA Received 5 June 2002; accepted 23 April 2003

It is not clear whether the same is true for ALL, especially with the poorer results for patients who have ALL with donor lymphocyte infusion (DLI). We examined the lymphocyte counts at 3 weeks (day +21) and at 1 month (day +30) after matched related donor allogeneic transplantations for ALL to determine the effect of lymphocyte recovery in ALL.

Materials and methods We retrospectively reviewed the database of patients who had undergone allogeneic BMT at our institution from 1982 to 1999 and identified 43 patients who had undergone matched related allogeneic BMT for ALL. Syngeneic transplantations and peripheral blood stem cell transplantations were not included. The data for days +21 and +30 leukocyte counts, both total and differential, were collected from the medical records. The group was heterogeneous for the type of GVHD prophylaxis and disease status at transplantation. Patients in their first remission were considered to have nonadvanced disease; all others had advanced disease. Patient characteristics are detailed in Table 1. All 43 patients survived at least 21 days post–transplantation and were divided into two groups according to absolute lymphocyte count (ALC) for analysis of the effect of the day +21 lymphocyte count on the risk of relapse. The risk was examined at several levels of ALC, namely 150  106/l, 175  106/l, 200  106/l, and 225  106/l. Similarly, the effect of ALC at day +30 was examined at 150  106/l, 175  106/l, 200  106/l, and 225  106/l for 42 patients for whom the ALC on day +30 was available.

Conditioning regimens The conditioning regimen used most frequently was cyclophosphamide (120 mg/kg) with fractionated total body irradiation (1320 cGy). The other main preparative regimen was etoposide (60 mg/kg) with total body irradiation.

GVHD prophylaxis The combination of cyclosporine and methotrexate was the prophylactic regimen used most often (Table 1). The cyclosporine dosage was 2.5 mg/kg twice daily on day –1; thereafter, 1.5 mg was given twice daily. The methotrexate dosage was 15 mg/m2 on day +1 and 10 mg/m2 on days +3, +6, and +11. The prednisone dosage was 0.5 mg/kg on days +8 to +14 and 1 mg/kg from days +15 to +28, tapered thereafter as indicated clinically. Acute GVHD was treated with methylprednisolone (1–2 mg/kg) as soon as the diagnosis was confirmed.

Effect of slow lymphocyte recovery after BMT S Kumar et al

1866 Table 1 Characteristics of patients with ALC>175  106/l and patients with ALCp175  106/l on day 21 (N = 43)a ALC>175  106/l (n = 28)

ALCp175  106/l (n = 15)

32 (9–51) 6

25 (12–47) 7

19 9

11 4

Disease status at transplantation Advanced Nonadvanced

11 17

10 5

HLA match Complete 1-Antigen mismatch

24 4

13 2

Conditioning regimen CY+TBI VP-16+TBI Other

15 11 2

13 2 0

GVHD prophylaxis CYA+MTX CYA+PRED CYA+MTX+PRED MTX Other

17 4 2 3 2

11 0 1 3 0

Characteristic Age (years) Time to transplantation after diagnosis (months) Male Female

ALC, absolute lymphocyte count; CY, cyclophosphamide; CYA, cyclosporine; GVHD, graft-versus-host disease; MTX, methotrexate; PRED, prednisone; TBI, total body irradiation; VP-16, etoposide. a Continous data are presented as median (range); categorical data as number of patients.

Supportive care All patients were treated in single rooms with high-efficiency particulate air filters. Standard precautions were observed for all patients with neutropenia. Fever with neutropenia prompted immediate use of a third-generation cephalosporin with the addition of vancomycin and amphotericin B sequentially over the next 48–72 h, according to the patient’s response to initial therapy. Antibiotic therapy was tailored further according to the results of blood cultures. Transfusions of red blood cells and platelets were given to maintain concentrations of hemoglobin greater than 8 g/dl and platelet counts of 10 000–20 000/mm3. Cytomegalovirus (CMV)-negative or leukocyte-reduced blood products were used for CMV-negative patient–donor pairs, and all cellular products were irradiated.

End points Relapse after transplantation was the primary end point. Remission and relapse were defined by conventional criteria. Treatment-related mortality was defined as death related directly to toxicity from the conditioning regimen or to other causes related to transplantation in patients free of disease or to both.

Statistical analysis The w2 test was used to compare nominal variables between the two groups; the t-test, to compare numerical variables. The Spearman rank correlation was used to evaluate the relationship between variables. The Kaplan–Meier product limit method was Leukemia

used for analysis of relapse-free survival and overall survival. Log-rank tests were used to compare survival curves. Patients were censored at the time of death (without relapse) or at the latest follow-up for estimating relapse-free survival. The Cox proportional-hazards model was used to compare the risk of relapse between the two groups. To analyze the effect of other variables on the risk of relapse, a multivariate analysis including age at transplantation, disease status (advanced or nonadvanced), HLA antigen match, and development of acute or chronic GVHD was performed. The study was approved by the Mayo Foundation Institutional Review Board.

Results The median follow-up for the group was 39 months (range, 1–198 months), with 40 patients surviving at least 100 days posttransplantation. In all, 16 patients relapsed after a median duration of 4.5 months; 14 of these 16 are dead and two were alive at latest follow-up. Of the remaining 27 patients, 11 have died of transplantation-related complications, GVHD, or late infections without evidence of recurrent disease. In total, 16 patients have been free of disease and were alive in continuous remission at a median follow-up period of 106 months (range, 18–198 months). The median ALC for the entire group on day 21 was 280  106/l (range, 40–920  106/l) and the median total white blood cell count was 2300  106/l (range, 100–6500  106/l) (Table 2). When the day +21 lymphocyte counts were analyzed, a trend toward a higher risk of relapse was present with lower ALC at all levels, and these differences were statistically significant at all levels (150  106/l, 175  106/l, 200  106/l, and 225  106/l). The most significant difference was seen at a level of 175  106/l. At this cutoff level, there were 15 patients (35%) in the slower recovery group (ALCp175  106/l) and 28 patients (65%) in the faster recovery group (ALC4175  106/l). Patients who had an ALC of 175  106/l or less at day +21 had a significantly higher risk of relapse than those with greater lymphocyte counts (relative risk, 4.06; 95% confidence interval, 1.47–11.2; P ¼ 0.0068). Log-rank comparison of the Kaplan– Meier survival curves demonstrated a significant difference in the relapse-free survival between the two groups (P ¼ 0.0028) (Figure 1). The 15 patients who had a slow recovery of their lymphocyte counts also had a lower overall survival than the other 28 patients (P ¼ 0.0275) (Figure 2).

Table 2 Post-transplantation results for patients with ALC>175  106/l and patients with ALCp175  106/l on day 21 (N = 43)a Characteristic Total white blood cell count at day 21 (  106/l) ALC at day 21 (  106/l) Alive at latest follow-up In continuous remission at latest follow-up Relapse Nonrelapse mortality Acute GVHD Chronic GVHD

ALC>175  106/l (n=28)

ALCp175  106/l (n=15)

500 (100–4700)

2750 (300–6500)

390 (180–920) 15 (54) 13 (46)

100 (40–170) 3 (20) 3 (20)

6 9 18 15

(21) (32) (64) (54)

10 2 12 7

(67) (13) (80) (47)

ALC, absolute lymphocyte count; GVHD, graft-versus-host disease. a Continuous data are presented as median (range); categorical data as number of patients (percentage of sample).

Effect of slow lymphocyte recovery after BMT S Kumar et al

1867 Table 3

Multivariate analysis of patient variables on risk of relapse

Variable 6

Day +21 ALCp175  10 /l Advanced disease No acute GVHD No chronic GVHD

RR

95% CI

P

4.5 1.79 0.367 12.1

1.24–16.6 0.49–6.53 0.09–1.5 2.9–49.9

0.022 0.38 0.162 0.0006

ALC, absolute lymphocyte count; CI, confidence interval; GVHD, graftversus-host disease; RR, relative risk.

Figure 1 Relapse-free survival for patients with an ALCp175  106/l and for those with an ALC4175  106/l at day 21 after matched related donor allogeneic transplantations for acute lymphoblastic leukemia (P ¼ 0.0028).

Figure 2 Overall survival for patients with an ALCp175  106/l and for those with an ALC4175  106/l at day 21 after matched related donor allogeneic transplantations for acute lymphoblastic leukemia (P ¼ 0.0275).

None of the levels of ALC at day +30 reliably predicted the risk of relapse. Also, the effect of the day +30 ALC on overall survival did not differ between the two groups at any level of the day +30 ALC. In the multivariate analysis (including disease status, HLA antigen match, development of acute or chronic GVHD, and ALC at day +21), patients with a day +21 ALC of 175  106/l or less showed a 4.5-fold greater risk of relapse than patients with a day +21 ALC greater than 175  106/l (P ¼ 0.022) (Table 3). The only other factor that was significant for the risk of relapse was the development of chronic GVHD, which appeared to be protective. No correlation existed between development of acute GVHD or chronic GVHD and lymphocyte recovery. There was no correlation between the time to neutrophil engraftment and the lymphocyte count on day 21 (r ¼ 0.276; P ¼ 0.09).

Discussion Therapeutic options are limited for patients who have a relapse post-transplantation because they tolerate further chemotherapy

poorly, and subsequent transplantations have limited efficacy. Limited success has been reported with aggressive chemotherapy after relapse in patients who have received allogeneic or autologous transplants for ALL.15 Infusion of peripheral blood lymphocytes from the same original donor (DLI) has been used as a salvage procedure for relapse and is aimed at harnessing the power of the graft-versus-leukemia (GVL) effect.16 Several reports have demonstrated long-term survival with DLI, with the greatest benefit in patients with chronic myelogenous leukemia.17–19 This approach tends to have less efficacy in patients with ALL than in patients with other leukemias (such as chronic myelogenous leukemia and acute myelogenous leukemia), with a 3-year relapse-free survival of 10–15%.20 Efforts have been made to evaluate its role in preventing relapse;21,22 however, DLI is also associated with a significant risk of acute and chronic GVHD. Other approaches for prevention of relapse have included the use of cytokines, such as interferon-a and interleukin-2 (IL-2), or rapid discontinuation of immunosuppressive therapy.17 As only certain patients have a relapse after allogeneic BMT, a need exists for prognostic variables that can prospectively identify patients at risk of relapse and thereby help select appropriate patients early for prophylactic immunotherapy or other interventions. Our data validate the importance of a rapid lymphocyte recovery on prevention of relapse after allogeneic BMT for ALL, similar to what we and others have observed in patients with acute myelogenous leukemia.13,14,23 The antileukemic effect of allogeneic stem cell transplantation is dependent on two components: (1) the conditioning chemotherapy contributing to tumor reduction by killing off chemosensitive cells and (2) the strong adoptive GVL response mediated by the immunocompetent cells in the stem cell graft, which eliminates minimal residual disease. The importance of the GVL effect has been known since its first description by the Seattle group in the 1970s, and has most recently been highlighted by the success of nonmyeloablative allogeneic transplants.24,25 The importance of each of these components is further highlighted by the increased relapse rates for patients with syngeneic transplants and autologous stem cell transplants. The decreased incidence of relapse among patients in whom acute GVHD develops after allogeneic transplantation for ALL also points toward the importance of the GVL effect.26,27 Current evidence suggests that donor-derived lymphocytes play a major role in eliminating the residual leukemic cells after allogeneic BMT.25,28–30 CD4 cells and CD8 cells,31,32 as well as natural killer (NK) cells,33,34 play a significant role in the genesis of the GVL reaction. Elevated numbers of activated T cells (DR+) have been noted in the early post-transplantation period.35,36 NK cells also appear to have an important role in the development of the GVL reaction, and their activity appears to be independent of the development of acute GVHD.37–39 The immunologically mediated destruction of malignant cells is likely to be most effective during the early post-transplantation period because of the minimal residual disease state present at Leukemia

Effect of slow lymphocyte recovery after BMT S Kumar et al

1868 that time. However, immune reconstitution as a whole, and lymphocyte recovery in particular, is often substantially delayed after BMT. After related marrow transplantations, the total lymphocyte count returns to normal by about 3 months posttransplantation.40,41 However, the total number of T cells, especially CD4+ T cells, remains low for up to 12 months posttransplantation (or longer in patients with chronic GVHD) in contrast to the number of CD8+ cells, which usually returns to normal quickly.34,42 During the early post-transplantation period, the number of NK cells increases rapidly, and NK cells constitute the majority of the peripheral blood mononuclear cells.43,44 NK cells are capable of mediating cytotoxicity without prior sensitization45 and may be responsible for most of the early antileukemic activity of the graft. Slow recovery of NK cells and CD8+ cells after allogeneic BMT has been correlated with an increased risk of relapse.34 Conceivably, patients in our study with slow lymphocyte recovery had decreased numbers of NK cells and T cells during the early posttransplantation period, thus depriving them of the GVL effect and leading to an increased risk of relapse. Unfortunately, the retrospective nature of our study precludes a detailed analysis of lymphocyte subsets, which would have been more informative in understanding some of the mechanisms behind this observation. Similar protective effects of a fast lymphocyte recovery have been observed in the context of autologous stem cell transplantation performed for various hematologic malignancies.46 Studies have shown that there is a rapid recovery of NK cells in the immediate post-transplantation period in patients undergoing autologous stem cell transplantation. These NK cells can be upregulated by IL-2 and interferon-a as shown by in vitro studies,47 a phenomenon that may occur in patients with allogeneic transplants as well. Early identification of patients at high risk of relapse is important because it could potentially allow intervention specifically aimed at prevention of relapse. Prevention could take the form of a rapid taper of immunosuppressive therapy with the goal of triggering a GVL reaction, use of DLI, or initiation of immunomodulatory biological agents, such as interferon or interleukins (eg, IL-2 or IL-12). IL-2 enhances the cytotoxicity of NK cells against various cell lines47–49 and decreases the relapse rate after T-cell-depleted allogeneic BMT.50 Interferon-a has had a favorable effect on relapse rates after allogeneic BMT.51 In the current study, there was a trend toward decreased overall survival among patients with slow recovery of lymphocyte counts. Similar observations have been made by Pavletic et al 23 among patients after allogeneic blood stem cell transplantation. In our study, an increased rate of relapse accounted for some of the excess deaths in the slow recovery group. However, infections and transplant-related toxicity also contributed to a decrease in overall survival in this group. Faster lymphocyte recovery may be a sign of more rapid immune reconstitution, leading to fewer infections and deaths. Decreased post-transplantation lymphocyte counts have been associated with CMV infection52 as well as with increased mortality from CMV disease.53

Conclusion We have shown that slow recovery of lymphocyte counts after allogeneic BMT for ALL is a predictor of post-transplantation relapse. Validation of this phenomenon for ALL is important especially because previous studies have indicated that GVHD Leukemia

has a less protective effect on relapse in patients with ALL than in those with acute myelogenous leukemia or chronic myelogenous leukemia. This information could potentially be combined with other known risk factors for relapse to better identify patients who are at high risk of early relapse after allogeneic BMT for ALL. Early identification of these patients enables early intervention with the immunotherapeutic options described above or enrollment of patients in trials that evaluate novel methods of relapse prevention. These principles probably apply to allogeneic peripheral blood stem cell transplantation as well;23 however, this finding needs to be verified by additional studies.

Acknowledgements We thank Ms Norine E Huneke for her expert assistance with BMT database management.

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